Hemoglobin has been previously shown to bind to carbonic anhydrase (EC 4.2.1.1) in vitro. The proposed research will employ four independent methods to measure the association constant for the binding of hemoglobin to carbonic anhydrase. The dependence of the physical interaction of these two proteins on physiological bariables will then be examined in order to assess the role of this interaction in the regulation oxygen and carbon dioxide transport by the red blood cell. These variables will include; (1) state of oxygenation of the hemoglobin, (2) organic phosphate interactions, (3) pH, (4) temperature and (5) membrane interactions. To complement the binding studies, the influence of hemoglobin on the steady state kinetics of carbonic anhydrase as well as the effect of carbonic anhydrase on hemoglobin oxygen dissociation will be determined. Comparative studies with evolutionary homologous hemoglobins and carbonic anhydrases, as well as with chemically modified proteins, will be included in order to characterize the binding domains, and provide insights into the molecular evolution of, the two proteins.